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. 2012 Nov 6;7(11):e48539. doi: 10.1371/journal.pone.0048539

Figure 1. HSP90 inhibition stimulates mature VEGFR2 degradation and blocks intracellular signaling in primary endothelial cells.

Figure 1

(A) HUVECs were subjected to geldanamycin (17-DMAG) treatment for indicated times, lysed and processed for immunoblotting (IB) to analyze indicated protein levels. Arrowhead indicates mature VEGFR2; TfR, transferrin receptor. (B) Quantification of mature VEGFR2 levels in control and geldanamycin-treated endothelial cells; data shown is representative of 3 independent experiments. (C) HUVECs were treated with dimethyl sulfoxide (DMSO; control) or geldanamycin (4 h) and stimulated with VEGF-A (5 min) followed by immunoblotting (IB) of phosphorylated and total levels of intracellular signaling enzymes. PLCγ1, phospholipase Cγ1; ERK1/2, extracellular signal-regulated kinase 1/2. (D) Immunoblot data from panel C were quantified for phosphorylated and activated VEGFR2-pY1175; error bars denote ±SEM (n≥3), ***p<0.005 using one-way ANOVA. (E) HUVECs were treated with geldanamycin (4 h) and the cell surface labeled with biotin on ice. VEGFR2 and transferrin receptor (TfR) were immunoprecipitated (IP) and analyzed by immunoblotting (IB) alongside whole cell lysates using streptavidin-HRP (Strept HRP; streptavidin-horsereadish peroxidase). HSC70, heat shock cognate protein chaperone. (F) Immunoblot data from panel E were quantified for mature VEGFR2; error bars denote ±SEM (n≥3), **p<0.01; ***p<0.005 using one-way ANOVA.